Introduction
Helicobacter pylori is the most common cause of chronic gastric infection in humans. Helicobacter pylori infection has been reported throughout the world, with a mean prevalence of 50 per cent, which increases with age. After entering the gastrointestinal system, H pylori colonises the gastric epithelial cell surface. Helicobacter pylori infection of the gastrointestinal system is a major factor in the development of chronic mucosal inflammation, and has also been identified as a carcinogen promoting gastric carcinoma. The outcome of infection depends on the bacterium's potential to pass through the physicochemical gastric barriers, and on its replication and further dissemination. To date, research has focussed on the virulence factors which enable colonisation, persistence, disease induction, and dissemination to other organs and organ systems.
Isolation of H pylori is not an easy task and is affected by various factors, including certain physicochemical and biological properties of the bacterium, and the type of diagnostic procedure used (i.e. invasive or non-invasive).
Non-invasive diagnostic methods detect the presence of H pylori by demonstrating specific bacterial activity (primarily via the activity of urease), or by identifying specific antibodies in the serum and/or saliva, or a specific antigen in the stool. The sensitivity and specificity of the 13C urea breath test exceeds 90 per cent. This test is effective in the diagnosis of H pylori in dyspeptic patients, and is also useful in monitoring the therapeutic outcome of H pylori eradication in patients with non-ulcerative dyspepsia and duodenal ulcer.Reference Logan, Lee and Megraud1, Reference Breuer and Graham2 Despite the high specificity and sensitivity of the urea breath test for H pylori, other urease-producing bacteria in the oral cavity and stomach can produce false positive results. So far, five bacterial species with urease activity have been isolated from the oral cavity and/or stomach: Proteus mirabilis, Citrobacter freundii, Klebsiella pneumoniae, Enterobacter cloacae and Staphylococcus aureus.Reference Osaki, Mabe, Hanawa and Ksmiya3
Recently, several molecular techniques have been introduced into the H pylori diagnostic procedure. The presence of H pylori DNA has been demonstrated in biopsy specimens of gastric mucosa and other tissues, using polymerase chain reaction analysis. This technique can also be used to demonstrate and identify various bacterial strains and subtypes.Reference Hua, Birac, Megraud, Lee and Megraud4–Reference Wang, Lim, Shen, Yang, Chen and Wang6
The present study aimed to assess the presence of H pylori in clinically healthy laryngeal mucosa, using urea breath testing and polymerase chain reaction analysis.
Materials and methods
This human study was appropriately reviewed and approved by the ethics committee of the Dr Josip Benčević General Hospital in Slavonski Brod, Croatia.
Twenty volunteers were randomly selected, and each gave written, informed consent.
The absence of pathological laryngeal changes was confirmed from the subjects’ medical history and clinical examination. The selected individuals had no history of chronic laryngitis, Reinke's oedema, vocal nodules, polyps or malignant tumours. In addition, they had not suffered any kind of gastrointestinal disease for the past three years. These subjects were scheduled for elective surgery for repair of bone fracture (eight subjects), pilonidal sinus (seven) or inguinal hernia (five). All subjects were men aged between 28 and 78 years (median, 53 years).
The 13C urea breath test was performed on two exhalation samples: the first obtained immediately after a test meal, using 75–100 mg of 13C- or 14C-labelled urea, and the second 30 minutes thereafter. If present in the host, bacterial urease converts urea to bicarbonate and ammonia ions. The 13CO2 or 14CO2 released by bicarbonate breakdown is then detected in the exhaled breath, giving a positive result.
All subjects were scheduled for elective operative procedures under general, endotracheal anaesthesia. Samples for H pylori DNA detection were obtained using the cytobrush technique, upon anaesthesia induction and immediately before intubation. After sampling, the brushes were immersed in Eppendorf microtubes containing 1.5 ml of sterile digestion buffer (50 mM Tris–HCl, pH 8.5, 1 mM ethylene diamine triacetic acid (EDTA) and 1 per cent Tween 20) and frozen at −20°C until analysis. Cells were removed from the brushes by vigorous shaking. Proteinase K was added to a final concentration of 100 µg/ml. Samples were incubated, with gentle shaking, for 10 hours at 37°C. The genomic DNA was extracted by two phenol chloroform extractions and precipitated at −20°C for 10 hours, with the addition of 1/10 volumes of 10 M ammonium acetate and 2.5 volumes of absolute cold ethanol, as previously described.Reference Mravak-Stipetic, Gall-Trošelj, Lukac, Kusic, Pavelic and Pavelic5 After a 10-minute centrifugation at 14 000 rpm at room temperature, the residual pellets were washed with 70 per cent ethanol, air-dried and dissolved in 30–50 µL Tris–EDTA buffer, pH 8.0. Nested polymerase chain reaction was performed as previously described.Reference Mravak-Stipetic, Gall-Trošelj, Lukac, Kusic, Pavelic and Pavelic5 All reactions were performed in triplicate, with positive and negative controls, according to standardised laboratory practice.
Results and analysis
The test results are summarised in Table I.
Table I Helicobacter pylori test results*
* In healthy laryngeal mucosa.†Total = 20. UBT = urea breath test; +ve = positive; −ve = negative; PCR = polymerase chain reaction
The 13C urea breath test revealed that eight (40 per cent) subjects were H pylori positive and 12 (60 per cent) were H pylori negative. Positive results were recorded in 25 per cent of subjects aged 50 years or less, and in 75 per cent of those older than 50 years.
Polymerase chain reaction analysis failed to detect the presence of H pylori DNA in any patient.
Discussion
The prevalence of H pylori infection exceeds 50 per cent of adults worldwide, and is considered a major cause of pathology. Helicobacter pylori has been primarily described as causing gastrointestinal infection, and the species has been classified as having slow pathogenicity. Helicobacter pylori actively modifies the host's immune response, enabling a sustained state of balance between bacterial replication and the host's defence mechanisms. This explains the relatively mild chronic inflammatory reaction characteristic of H pylori chronic gastritis (prevalence, 60–100 per cent) and H pylori duodenal ulceration (prevalence, 90–100 per cent).Reference Kuipers, Thijs and Festen7, Reference Genta, Gurer and Graham8 In addition, H pylori has been identified as a significant carcinogen for gastric carcinoma.Reference Hunt9
Given the evidence of high H pylori seroprevalence and infection rates, this organism may also play a role in the aetiology of extra-gastrointestinal diseases such as respiratory disorders (e.g. chronic obstructive pulmonary disease, bronchiectasis, lung cancer, pulmonary tuberculosis and bronchial asthma), vascular disorders (e.g. ischaemic heart disease, stroke, primary Raynaud's phenomenon and primary headache), autoimmune disorders (e.g. Sjögren's syndrome, Henoch–Schönlein purpura, autoimmune thrombocytopenia, autoimmune thyroiditis, Parkinson's disease, idiopathic chronic urticaria, rosacea and alopecia areata) and other conditions (e.g. iron deficiency anaemia, growth retardation, liver cirrhosis and nasal polyposis).Reference Whincup, Mendall, Perry, Strachan and Walker10–Reference Prelipcean, Mihai, Gogắlniceanus, Mitricắ, Drug and Stanciu13
Helicobacter pylori colonisation has been detected in the healthy oral cavity and also in the presence of various types of oral pathology, including ulcerative stomatitis, atrophic glossitis, dental plaque and chronic tonsillitis.Reference Koc, Anikan Osman, Atasoy and Aksoy12, Reference Karczewska, Konturek, Konturek, Cześnikiewicz, Sito and Bielański14, Reference Gall-Trošelj, Mravak-Stipetić, Jurak, Ragland and Pavelić15 Despite numerous studies on the route of H pylori colonisation, the role of H pylori in the oral cavity remains obscure. Is H pylori part of the normal oral microflora, or is it only transiently present in the oral cavity? Gall-Trošelj et al. have demonstrated that there is no preferential site for H pylori colonisation in healthy oral cavity mucosa. On the other hand, mucosal changes may make the environment more acceptable for such colonisation.Reference Gall-Trošelj, Mravak-Stipetić, Jurak, Ragland and Pavelić15Helicobacter pylori reaches the oral cavity via occasional reflux from the gastric reservoir; it can also be introduced via oral–oral or faecal–oral transmission, and then swallowed into the stomach.Reference Nguyen, Engstrand, Genta, Graham and El-Zaatari16Helicobacter pylori has been commonly detected in the oral cavity of patients with dyspeptic symptoms, but gastric reinfection does not occur in these patients despite persistent oral H pylori colonisation.Reference Karczewska, Konturek, Konturek, Cześnikiewicz, Sito and Bielański14 A possible explanation is that these patients had only a low level of H pylori organisms in their oral cavity, which does not reach the critical threshold that would make colonisation of the stomach possible. However, in a more favourable environment the number of viable bacteria may increase to a level sufficient to cause gastric infection or reinfection.Reference Liu, Yue, Li, Wang, Jiang and Zhang17
• This study assessed the presence of Helicobacter pylori in normal laryngeal mucosa
• Polymerase chain reaction analysis and 13C urea breath testing were used
• These tests gave 0 per cent and 40 per cent positivity, respectively
• Helicobacter pylori is not a normal commensal in healthy laryngeal mucosa
Helicobacter pylori has a unique way of adapting to the gastric environment. It penetrates through the mucous layer to infect gastric epithelial cells, and produces enzymes which break down substances in the gastric juice. The most important of these enzymes is urease, which converts urea from saliva and gastric juice into bicarbonate and ammonia. The by-product, carbon dioxide, is then absorbed into the circulation and exhaled.Reference Artiko, Obradović, Petrović, Davidović, Grujić-Adanja and Nastić-Mirić18
We believe that there are other H pylori transmission modes besides the oral route, and that healthy laryngeal mucosa may be one of these. Our study aimed to assess the presence of H pylori in healthy laryngeal mucosa, by testing 20 individuals without laryngeal pathology and with no gastrointestinal disease over the previous three years. Despite variable 13C urea breath test results, polymerase chain reaction analysis showed that these subjects’ laryngeal mucosa was free of H pylori. These negative polymerase chain reaction results suggest that normal laryngeal mucosa acts as a barrier to H pylori infection. However, impaired laryngeal mucosa has been shown to be a favourable medium for H pylori colonisation.
Recently published findings have suggested the importance of H pylori infection in chronic laryngeal mucositis.Reference Borkowski, Sudhoff and Koslowski19 However, Akbayir et al. failed to detect H pylori in carcinomatous and benign lesions of the laryngeal mucosa, using histology and immunohistochemistry.Reference Akbayir, Basak, Seven, Sungum and Erdem20 Fang et al. reported laryngeal H pylori colonisation in patients with vocal polyps, but concluded that the role of H pylori as an aetiological factor for vocal polyps remained inconclusive.Reference Fang, Lee, Li, Yang and Huang21 Rubin et al. found an increased titre of H pylori antibodies in patients with chronic laryngeal dysplasia and in those with head and neck carcinoma; they concluded that the presence of gastroesophageal reflux was a significant pathological process which may cause laryngopharyngeal bacterial colonisation, and which may be a cofactor in the development of various inflammatory processes that may be a basis for carcinogenesis.Reference Rubin, Benjamin, Prior and Lavy22 Titiz et al. used polymerase chain reaction methodology to identify H pylori in laryngeal carcinoma and mucosal tissue; they obtained positive results in 80.9 per cent of their cancer patients, but in none of their patients with benign mucosal neoplasias (e.g. polyps and nodules).Reference Titiz, Ozcakir, Ceyhan, Yilmaz, Unal and Akyon23 However, Cvorovic et al. detected H pylori in the nasal polyps of patients who also had H pylori in their stomach.Reference Cvorovic, Brajovic, Strbac, Milutinovic and Cvorovic24 Zhuo et al. conducted a meta-analysis of 15 published studies on the impact of H pylori on the development of invasive laryngeal carcinoma.Reference Zhuo, Wang, Zhuo and Zhang25 Finally, Grbesa et al. reported that H pylori can be present in laryngeal squamous cell carcinoma, but that its presence does not seem to influence the IGF2 (Insulin like growth factor)/H19 (gen for a long noncoding RNA, this gen seems to have a role in some forms of Cancer) imprinting status.Reference Grbesa, Marinkovic, Ivkic, Kruslin, Novak-Kujundzic and Pegan26
Acknowledgement
This work received financial support from Dr Josip Benčević General Hospital, Slavonski Brod, Croatia.
